Affinity-based immunoassays, due to their sensitivity, are routinely used to detect and measure the presence and the concentration of an analyte in a sample. The analyte may be any of the wide variety of materials, such as drugs, pollutants, chemicals, contaminants, or the like. The most common approach for performing detection and quantification of analytes with low molecular mass (<1000) is the competitive immunoassay. This assay, which exists in multiple formats, involves a target analyte, a specific receptor that binds to the analyte, and a corresponding analyte-conjugate, which consists of a target analyte (or a derivative of the target analyte) conjugated to a detectable label such as an enzyme.
Competitive assays are known to exhibit a nonlinear dose-response curve, the shape of which is dictated by the mass-action principle and the affinity of the receptor for the analyte and analyte-conjugate. The dynamic range offered by such assays is often limited to approximately one order of magnitude when using monoclonal antibodies as receptors. Unfortunately, the clinically relevant range of concentrations of a large number of important analytes can be much larger than an order of magnitude, thereby exceeding the measurable range of most competitive assays.
The most straightforward technique to extend the dynamic range of an assay is to dilute the sample and rerun the assay. This method preserves the accuracy of the assay and increases the dynamic range. Such a strategy might be reasonable for clinical settings that perform screening, in which analyte concentrations are usually distributed over a very limited range of concentrations. In such cases, the probability of needing dilution is very low and therefore infrequent. Clinics that perform high-throughput screening are particularly suited towards dilution as a means of extending the assay dynamic range since the time and cost associated with sample dilution is relatively inconsequential.
However, there are many other non-screening clinical settings in which samples can exist with a very broad distribution of analyte concentrations, making it necessary to dilute samples frequently. For example, methadone maintenance clinics (that monitor the compliance of drug addicts under therapy for their addiction) require the frequent and accurate testing of a population of patients with wide range of drug concentrations. If dilution is used to provide the needed assay dynamic range, considerable cost can result; both in terms of the cost of additional reagents and also as a result of the longer time interval before a result is obtained. Smaller clinics are particularly sensitive to the increased cost associated with dilution since they cannot benefit from the same economies of scale that the high throughput clinics enjoy.
U.S. Pat. No. 5,585,241 and WO8911101 describe assay techniques that utilize high and low affinity receptors, respectively, coated onto different types of monodisperse particles. However, these methods require the production of two different types of receptors for the analyte, a task that requires long development times. Furthermore, it is difficult to satisfy the requirement for highly controlled receptor affinity over multiple batches of reagents. U.S. Pat. No. 6,551,788 provides assays having a wide dynamic range by simultaneously incubating a sample with two or more independently determinable classes of a receptor coated particles differing from each other in size. Again, this method requires the costly and onerous task of preparing dedicated reagents with a high degree of control.
What is therefore needed is an assay method that offers an extended dynamic range in a simple, cost-effective and practical method without requiring a separate assay and the consumption of additional reagents.